KHO ST S C CH ENZYME

ACETYLCHOLINESTERASE
BNG SC K LP MNG
I HC QUC GIA THNH PH H CH MINH
TRNG I HC KHOA HC T NHIN
KHOA HA HC B MN HA HU C
SVTH: Nguyn nh T
GVHD: GS.TS. Nguyn Kim Phi Phng
Ti:
1
2
3
http://www.google.com.vn/search?q=alzheimer&hl=vi&prmd=imvnsb&source=lnms&tbm=isch&sa=X&ei=9GnoT4-
cFM2ciAf6rLVZ&sqi=2&ved=0CE8Q_AUoAQ&biw=1280&bih=675
4
http://upload.wikimedia.org/wikipedia/commons/8/89/AChe_mechanism_of_action.jpg (24/06/2012)
5
arka tpnkov
*,a
and Karel Komers
b
, (2008). Cholinesterase and cholinesterase Inhibitors. Current Enzymee Inhibition. 4, 160-171.
Susana Lo1pez, Jaume Bastida, Francesc Viladomat, Carles Codina*, (2002). Acetylcholinesterase inhibitory activity of some Amaryllidaceae alkaloids and
Narcissus extracts. Life Sciences. 71, 2521-2529.
IC
50
= 1 M
IC
50
= 0.1 M
IC
50
= 1.61 M
IC
50
= 9.6 M
IC
50
= 152.32 M IC
50
= 3.87M IC
50
= 47.21 M
6
C lp t cy
Huperzine serrate
C lp t cy
Buxus hyrcana
Dae Keun Kim, Ki Taek Lee, Nam-In Baek
1
, Sung-Hoon Kim
2
, Hee Wook Park, Jong Pil Lim, Tae Yong Shin, Dong Ok Eom, Jae Heon Yang, and Jae Soon
Eun, (2004). Acetylcholinesterase inhibitors from the aerial parts of Corydalis speciosa. Archibes of Pharmacal Research. 27(11), 1127-1131.
M. Iqbal Choudhary
a)b)*
, Salma Shahnaz
a)
, ShehnazParveen
a)
, Asaad Khalid
a)
, M. Ahmed Mesaik
b)
, S. Abdul Majeed Ayatollahi
c)
,and Atta-ur-Rahman
a)b)
,
(2006). New cholinesterase-inhibiting triterpenoid alkaloids from Buxus hyrcana. Chemistry and Biodiversity. 3, 1039-1051.
IC
50
= 89 M
IC
50
= 16.1 M IC
50
= 5.8 M IC
50
= 3.3 M
IC
50
= 145 M
IC
50
= 468 M
IC
50
= 310 M
IC
50
= 83 M
IC
50
= 210 M
IC
50
= 83 m175.4
C lp t Corydails speciosa
arka tpnkov
*,a
and Karel Komers
b
, (2008). Cholinesterase and cholinesterase Inhibitors. Current Enzymee Inhibition. 4, 160-171.
8
Nhc im:
Cht phn tch phi km
bay hi.
Hiu qu tch cht nh
lng v tch lng mu
ln khng cao
Nguyn Kim Phi Phng, Phng Php C Lp Hp Cht Hu C. i Hc Quc Gia Thnh Ph H Ch Minh, (2007).
http://upload.wikimedia.org/wikipedia/commons/4/41/TLC_black_ink.jpg
u im:
Cn lng mu t,nhanh
C th phn tch ng thi
mu v cht i chng
Dng c n gin
Kch thc mu ln c th
kim sot c.
9
KHO ST S C CH
ACETYLCHOLINESTERASE
BNG SC K LP MNG
[7] A. Marston, J. Kissling and K. Hostettmann, (2002). A Rapid TLC Bioautographic Method for the detection of Acetylcholinesterase and Butyrylcholinesterase
inhibitors in plants. Phytochem. Anal. 13,51-54.
[8] In Kyung Rhee
*
, Michiel van de Meent, Kornkanok Ingkanianan, Robert Verpoorte, (2001). Screening for acetylcholinesterase inhibitors from Amaryllidaceae
using silica gel thin-layer chromatography in combination with bioactivity staining. Journal og Chromatography A. 915, 217-223
10
[7] A. Marston, J. Kissling and K. Hostettmann, (2002). A Rapid TLC Bioautographic Method for the detection of Acetylcholinesterase and Butyrylcholinesterase
inhibitors in plants. Phytochem. Anal. 13,51-54.
Trng
Mu
11
(1)
(2)
Trng Mu
i chng
dng
[7] A. Marston, J. Kissling and K. Hostettmann, (2002). A Rapid TLC Bioautographic Method for the detection of Acetylcholinesterase and Butyrylcholinesterase
inhibitors in plants. Phytochem. Anal. 13,51-54.
Mu vi nng
1 mg/ml
Mu gii ly
trn bn mng
Quan st cc vt trn bn mng
- Phun enzyme AChE
Enzyme 1000 U
150 mg BSA
150 ml tris-HCl 0.05 M
-Phun thuc th mui fast blue B
40 ml mui fast blue B 2.5 mg/ml
10 ml naphthyl acetate 2.5 mg/ml
- Sy kh bn mng
bn mng trong
20 pht 37
0
C
- Sc k bn mng, gii ly trong
h CHCl
3
-MeOH-H
2
O (65:35:5)
Trng Mu
i chng
dng
Kt qu
[7] A. Marston, J. Kissling and K. Hostettmann, (2002). A Rapid TLC Bioautographic Method for the detection of Acetylcholinesterase and Butyrylcholinesterase
inhibitors in plants. Phytochem. Anal. 13,51-54.
13
Dch chit cao ethanol 80%:
(1) Galanthamine
(2) Galanthus nivalis
(3) Leucojum vernum
(4) Narcissus pseudonarcissus
(5) Chelidonium majus
(6) Ismene festalis
(7) Nerine bowdenii
(8) Crinum powellii
(9) Sprekelia formosissima
R
f
l
1 2 3 4 5 6 7 8 9
R
f
0
g
Kt qu
14
Galanthamine
Physostigmine
g
Dung mi gii li: CHCl
3
-MeOH-H
2
O (65:35:5)
1 0.1 0.01 0.001 0.0001
R
f
l
R
f
0
0.1 0.01 0.001 0.0001 0.00001
R
f
0
R
f
l
g
[8] In Kyung Rhee
*
, Michiel van de Meent, Kornkanok Ingkanianan, Robert Verpoorte, (2001). Screening for acetylcholinesterase inhibitors from Amaryllidaceae
using silica gel thin-layer chromatography in combination with bioactivity staining. Journal og Chromatography A. 915, 217-223.

Trng
Mu
15
(1)
(2)
(ATCI)
(DTNB)
Trng Mu
i chng
dng

[8] In Kyung Rhee
*
, Michiel van de Meent, Kornkanok Ingkanianan, Robert Verpoorte, (2001). Screening for acetylcholinesterase inhibitors from Amaryllidaceae
using silica gel thin-layer chromatography in combination with bioactivity staining. Journal og Chromatography A. 915, 217-223.
Mu vi nng
10 mg/ml
Mu gii ly
trn bn mng
-Sc k bn mng, gii ly
trong h CHCl
3
-MeOH (8:2)
Quan st bn mng
-Phun enzyme AChE
Enzyme 1000 U
150 mg BSA
150 ml tris-HCl 0.05 M
-Phun thuc th Ellman
1 mM DTNB
1 mM ATCI
Sy kh bn
mng 3-5 pht
Trng Mu
i chng
dng
16
1, Hymanocallis festails Zwanenburg
2, Chlidanthus fragrans Herb
3, Narcissus Avalanche (Tazetta)
4, Nerine bowdenii
5, Narcissus Grand Soleil dOr (Tazetta)
6, Zephyranthes candida (Lindl) Herb
7, Crinum powellii Baker
8, Polianthes tuberosa L
9, Amaryllis belladonna L
10, Eucharis amazonica Linden ex Planch
11, Hippeastrelia
12, Habranthus robustus Herb. Ex Sweet
13, Rhodophiala bifida (Herb) Traub
14, Hymencallis Sulphur Queen
15, Sprekelia formosissima (L.) Herb)
Ganlathamine.
[8] In Kyung Rhee*, Michiel van de Meent, Kornkanok Ingkanianan, Robert Verpoorte, (2001). Screening for acetylcholinesterase inhibitors from Amaryllidaceae
using silica gel thin-layer chromatography in combination with bioactivity staining. Journal og Chromatography A. 915, 217-223.
Cao toluene
Cao methanol
17
H gii ly CHCl
3
MeOH (8:2)
[8] In Kyung Rhee
*
, Michiel van de Meent, Kornkanok Ingkanianan, Robert Verpoorte, (2001). Screening for acetylcholinesterase inhibitors from Amaryllidaceae
using silica gel thin-layer chromatography in combination with bioactivity staining. Journal og Chromatography A. 915, 217-223.

Quy trnh 1
Mu vi nng
10 mg/ml
Mu gii ly
trn bn mng
-Sc k bn mng, gii ly
trong h CHCl
3
-MeOH (8:2)
Quan st cc vt trn bn mng
-Phun enzyme AChE
Enzyme 1000 U
150 mg BSA
150 ml tris-HCl 0.05 M
-Phun dd thuc th Ellman
1 mM DTNB
1 mM ATCI
Sy kh bn
mng 3-5 pht
18
Quy trnh 2
-Phun dd DTNB 1 mM
- Sy kh bn mng
Mu vi nng
10 mg/ml
Mu gii ly
trn bn mng
-Sc k bn mng, gii ly
trong h CHCl
3
-MeOH (10:1)
-Phun dd thiocholine
Enzyme gc 3 U/ml
ATCI 1 mM
Sy kh bn
mng 3-5 pht
Quan st cc vt trn bn mng
trng mu
1
mu
2
i chng
[8] In Kyung Rhee
*
, Michiel van de Meent, Kornkanok Ingkanianan, Robert Verpoorte, (2001). Screening for acetylcholinesterase inhibitors from Amaryllidaceae
using silica gel thin-layer chromatography in combination with bioactivity staining. Journal og Chromatography A. 915, 217-223.
trng mu
1
mu
2
i chng
Quy trnh 1
Quy trnh 2
19
(ATCI)
(DTNB)
Hp cht
Nng
[mM (%)]
Bn silica gel Bn nhm oxid
c ch do
phn ng
ha hc
c ch do
enzyme
c ch do
phn ng
ha hc
c ch do
enzyme
Heptanal 720 (10%) + + + +
Decanal 530 (10%) + + + +
Cinanamaldehyde 790 (10%) + + + +
Anisaldehyde 820 (10%) + + + +
Benzaldehyde 100 + + - -
4-dimethylaminobenzaldehyde 100 - + - +
3-etoxy-4-
hydroxybenzaldehyde
100 - + - +
Diethylamine 960 (10%) - + - +
Triethylamine 717 (10%) - + - +
Ethanolamine 1670 (10%) - - - -
Triethanolamine 750 (10%) - + - +
Hexylamine 756 (10%) + + + +
Tryptamine 50 + + + +
Tyramine 50 - + - +
Galanthamine 1 - + - +
Physostigmine 1 - + - +
[8] In Kyung Rhee*, Michiel van de Meent, Kornkanok Ingkanianan, Robert Verpoorte, (2001). Screening for acetylcholinesterase inhibitors from Amaryllidaceae
using silica gel thin-layer chromatography in combination with bioactivity staining. Journal og Chromatography A. 915, 217-223.
20
[8] In Kyung Rhee*, Michiel van de Meent, Kornkanok Ingkanianan, Robert Verpoorte, (2001). Screening for acetylcholinesterase inhibitors from Amaryllidaceae
using silica gel thin-layer chromatography in combination with bioactivity staining. Journal og Chromatography A. 915, 217-223.
Toluene (1), hexane (2), chloroform (3), methanol 50% (4)
Hnh A. Hin hnh vi
thuc th anisaldehyde.
Hnh B. Quy trnh 1 Hnh C. Quy trnh 2
H dung mi CHCl
3
MeOH (10:1)
21
Kt qu th nghim cc cao th toluene t Nerine bowdenii
p dng mu
v cht chun
Pht trin bn
mng
Xc nh vt, qut, ph
Bung
gii li
My qut
Xut d liu hnh nh
k thut s
Linomat 5
applicator
[11] ManMohan Srivastava, (2011). High-Performance Thin-Layer Chromatography (HPTLC). Springer-Verlag Berlin Heidelberg. 44.
20 ng
0.013 ng
0.2 ng
0.8 ng
0.2 ng
22
TNG KT
Qua nghin cu c ch AChE bng TLC vi hai thuc th mui fast
blue B v Ellman th thuc th Ellman c th xc nh c cc hp
cht cho kt qu dng tnh gi.
a s cc hp cht c hot tnh c ch AChE u cha nitrogen.
23
[10] Christel Weins*, Hellmut Jork1, (1996). Toxicological evaluation of harmful substances by in situ enzymeatic and biological detection in high-performance
thin-layer chromatography. Journal of Chromatography A. 750, 403-407.
arka tpnkov
*,a
and Karel Komers
b
, (2008). Cholinesterase and cholinesterase Inhibitors. Current Enzymee Inhibition. 4, 160-171.
Alkaloid l hp cht c cha nitrogen d vng hin
din kh nhiu trong thc vt.
Cy c xem c alkaloid phi cha t nht 0.05 %
alkaloid so vi mu cy kh.
thun li, th cc nh khoa hc chia alkaloid
thnh 3 loai: alkaloid tht, protoalkaloid (phn t
khng c mch vng m c mch nhnh c cha
nguyn t nitrogen), alkaloid gi.
Trong t nhin cc alkaloid tn ti di dng cht
rn kt tinh ca mui cc acid hu c hoc v c, rt
t tn ti th lng tr nicotin
Tnh cht: c tnh baz yu v ty thuc s hin din
ca nhm th R
Alkaloid
Nguyn Kim Phi Phng, Phng Php C Lp Hp Cht Hu C. i Hc Quc Gia Thnh Ph H Ch Minh, (2007)
Bt cy
Bt loi bo
B cy, b Dch chit CHCl
3

Dch chit CHCl
3
m c
Dch CHCl
3
Dch chit nc
Dch chit nc
Dch chit nc Dch CHCl
3

Dch CHCl
3
Dch nc, b
Alkaoid c tnh
baz manh
Alkaoid c tnh
baz trung bnh
Dch CHCl
3

Alkaoid c tnh
baz yu
- Chit vi hexane hoc eter du ha loi bo
-kim n pH= 10-12
-Chit trong Soxhlet
- ui bt dm
- Chit vi dd nc acid 2%
(hp cht hu c
khc)
-Dd kim pH=6-7
-Chit bi CHCl
3

-Lm khan nc
-Thu hi dm
-Lm khan nc
bng Na
2
SO
4

-Thu hi dm
-Lm khan nc
-Thu hi CHCl
3

-Dd kim pH=10-12
-Chit bi CHCl
3

-dd kim pH= 2-3
-tn chit vi CHCl
3

Quy trnh tch chit alkaloid ra khi bt cy bng dm hu c
Nguyn Kim Phi Phng, Phng Php C Lp Hp Cht Hu C. i Hc Quc Gia
Thnh Ph H Ch Minh, (2007).
C 3 thuc th thng dng nhn danh alkaloid:
Mayer, Dragendoff, Wagner.
Thuc th Mayer: HgCl
2
+ KI + nc---------> ta
trng hoc vng.
Thuc th Dragendorff: Bi(NO
3
)
3
+HNO
3
+ KI +
HCl-----------------------> ta mu cam - nu.
Thuc th Wagner: I
2
+ nc ---------------> ta nu
dd acid long
cha alkaloid
dd acid long cha alkaloid
dd acid long cha alkaloid
- Phun thuc th Ellman
- Sc k bn mng, gii ly trong
h CHCl
3
-MeOH (8:2)
Mu vi nng
10 mg/ml
- Phun dung dch enzyme AChE
Mu gii ly trn
bn mng
Sy kh bn mng
3-5 pht
Quan st cc vt
trn bn mng
- Phun dd thiocholine
Enzyme gc 3 U/ml
ATCI 1 mM

- Sc k bn mng, gii ly trong
h CHCl
3
-MeOH (8:2)
Mu vi nng
10 mg/ml
- Phun dung dch DTNB 1 mM
- Sy kh bn mng
Mu gii ly trn
bn mng
Sy kh bn mng
3-5 pht
Quan st cc vt
trn bn mng
- Phun enzyme AChE
- Sc k bn mng, gii ly
trong
h CHCl
3
-MeOH (8:2)
bn mng trong
20 pht 37
0
C
Mu gii ly trn
bn mng
Mu vi nng
1 mg/ml
Quan st cc vt
trn bn mng
- Phun dung dich thuc th
mui fast blue B
Cao toluene ca N. bowdenii
Dch hexane Dch nc
hexane : nc
Dch hexane Dch methanol 50 % Dch chloroform Dch nc
methanol 50 % chloroform
Bung
gii li

My qut
Mu v cht chun Sc k lp mng chn lc
Ra bn mng
Pht trin bn mng

Chun b bn mng
Xc nh vt, qut, ph
p dng mu v cht chun
Xut d liu hnh nh
k thut s
Linomat 5
applicator